Mineralized osteoblast-derived exosomes and 3D-printed ceramic-based scaffolds for enhanced bone healing: A preclinical exploration

In regenerative medicine, addressing the complex challenge of bone tissue regeneration demands innovative strategies. Exosomes, nanoscale vesicles rich in bioactive molecules, have shown great promise in tissue repair. This study focuses on exosomes derived from mineralized osteoblasts (MOBs), which play a pivotal role in bone formation. We investigated the therapeutic potential of exosomes isolated from osteoblasts cultured in osteogenic medium for 21 days, delivered via 3D-printed gyroid scaffolds composed of hydroxyapatite (HA) and tricalcium phosphate (TCP). The exosomes were characterized through nanoparticle tracking analysis to determine size, morphology, and concentration, while proteomics revealed their cargo contents. In vitro, rabbit bone marrow mesenchymal stromal cells (rBMSCs) were cultured as monolayers and within ceramic scaffolds, where MOB-derived exosomes were shown to promote osteogenic differentiation. In vivo, their osteoconductive and bone augmentation capabilities were evaluated in two rabbit calvarial models, while the osteoinductive potential was further tested in a heterotopic mouse model. Neo-bone formation was assessed using µCT and histological analysis. Our findings demonstrated that MOB-derived exosomes upregulated bone-related gene expression and promoted mineralization in rBMSCs, even in the absence of osteogenic medium. Proteomics confirmed the presence of bone-associated proteins in these exosomes. In rabbit models, however, exosomes did not significantly enhance bone formation. In contrast, in the heterotopic mouse model, exosomes functionalized onto ceramic scaffolds exhibited strong osteoinductive activity. This study highlights the potential of MOB-derived exosomes to enhance 3D-printed ceramic scaffolds for bone regeneration, offering a promising avenue for bone healing without the need for additional growth factors or stem cells. STATEMENT OF SIGNIFICANCE: This study presents a novel strategy for bone regeneration by integrating exosomes derived from mineralized osteoblasts (MOBs) into 3D-printed ceramic scaffolds. While exosomes are increasingly studied for regenerative applications, the unique osteoinductive potential of MOB-derived exosomes has remained underexplored. Our findings demonstrate that these exosomes promote osteogenic differentiation and mineralization of stem cells without added growth factors. In vivo, their efficacy varied by anatomical site and species, highlighting key considerations for clinical translation. This work introduces a cell-free, bioactive scaffold platform that leverages naturally secreted vesicles for bone repair, offering potential alternatives to cell-based therapies. It will be of broad interest to researchers in tissue engineering, biomaterials, and translational regenerative medicine.